Induction of Xylanase and Cellulase Genes from Trichoderma harzianum with Different Carbon Sources

MATERIALS AND METHODS

Selection of fungi: A fungal strain Trichoderma harzianum (E-58) strain was employed to check the induction of xylanase and cellulase genes using different carbon sources. The fungus was obtained from National Institute for Biotechnology and Genetic Engineering (NIBGE) Faisalabad.

Substrates: Different substrates (carboxymethycellulose (CMC), xylan and glucose) were used for the induction of the cellulose xylanase genes from Trichoderma harzianum.

Media and culture conditons

Inoculum: Flasks were autoclaved at 121 °C for 15 minutes and inoculated with a loopful of fungal spores suspended in 100 mL of Vogel’s medium. The flasks were incubated at 28°C. The incubation time was limited to 24 h with shaking at 120 rpm in orbital shaker.

Enzyme assays
Xylanase assay: Activity of the enzyme was checked by Miller method (1959). One mL citrate phosphate buffer, 0.5 mL Xylan (1%) and 0.5 mL of diluted enzyme mixture were incubated at 50°C for 30 min. After expiry of time 3 mL DNS reagent was added and mixture was placed in boiling water bath for 5 min and the absorbance was noted at 550 nm. The international units of xylanase produced per mL were calculated by factor obtained from standard curve of xylose (Fig. 1).

Exoglucanase assay: Exoglucanase assay is based on the fact that the enzyme releases free cellobiose from avisel. One milliliter of diluted enzyme extract was incubated with 1 mL of 0.1 mL citrate buffer at pH 4.8 at 40^oC for 30 min. The reaction was stopped by boiling for 15 minutes and cooling in ice. After cooling, absorbance was measured at 540 nm. The international units of exoglucanase produced mL^–1 were calculated by factor obtained from the standard curve of cellobiose (Fig. 2).

Fig. 1:	Standard curve of xylose

Fig. 2:	Cellobiose standard curve

Fig. 3:	Standard curve of glucose

Fig. 4:	Standard curve of p-nitrophenol

Endoglucanase assay: It was performed as described by Gadgil (1995). Briefly 1 mL of appropriately diluted enzyme (0.01mL) was incubated for 30 minutes with 1 mL of 1% CMC and 1 mL of 0.1M citrate buffer of pH 4.8 at 50^oC. The reaction was trimmed by adding 3 mL of DNS reagent and boiled for 15 min and cooled in ice. The international units of endoglucanase produced mL^–1 were calculated by factor obtained from standard curve of glucose (Fig. 3).

β-glucosidase assay: It is based on the principal that p-nitrophenyl β-D-glucopyranoside is hydrolyzed by β-glucosidase to yield β-D-glucose and p-nitrophenol. One mL of β-D-glucopyranoside solution (substrate) in buffer and 1 mL of appropriately diluted enzyme were added and incubated at 40^oC for 10 min. Three mL of 1M Na₂CO₃ (Stopper) were added and volume was made up to 10 mL with distilled water. Absorbance was measured at 400 nm. The international units of β-glucosidase produced mL^–1 were calculated by factor obtained from standard curve of p-nitrophenol (Fig. 4).

RESULTS AND DISCUSSION

Growth of Trichoderma harzianum:The fungus Trichoderma harzianum was employed in this research. Earlier Esterbauer et al. (1991) found that Trichoderma strains are preferred microorganisms for the production of complete cellulases. Wong and Saddler (1992) studied properties and applications of xylanases from Trichoderma spp, which produced these enzymes with high xylanolytic activity. They isolated and purified different xylanases form Trichoderma species.

EPM (enzyme production medium) was used for the growth of fungus but growth did not come out. However Ulhoa et al. (1991) obtained positive results from EPM for the growth of Trichoderma harzianum for large-scale production of chitobiase. MYG medium (Heesing et al., 1994) was tried for sporulation of the Trichoderma harzianum in agar slants but desirable results could not be achieved. After failing to get the growth of Trichoderma harzianum on EPM and MYG media, Vogel’s medium was used for the growth of Trichoderma harzianum. Spores of T.harzianum were found on agar slants containing 0.2% xylan or CMC. The spores were inoculated in shake culture. T. harzianum was grown on Vogel’s medium for 5 days at 120 rpm.

Optimum pH for Trichoderma harzianum: The pH of the Vogel’s medium was adjusted to 5.5 to obtain the maximal production of the xylanases and cellulases from Trichoderma harzianum. Earlier Simpson (1991) worked on the production of thermostable endo 1-4 β xylanase from culture supernatant of Thermogota spp and noted that the optimum pH was between 5.0 and 5.5 for its activity. Rose and Van (2002) found that xylanase enzyme expresses its highest activity at pH 5-6. Similarly De Paula-Silveira et al. (1999) found that xylanase from T. harzianum had a maximum activity at pH 5.0. Milagres et al. (1993) worked on the production and characterization of xylanase from Penicillum Janthium and found that optimal pH for xylanase activity was 5.5.

Srivastava et al. (1984) used cellobiose and 4 nitro phenyl β-D glucosidase as substrate for the growth of Aspergillus Wentii to study the kinetic characteristics of β-D glucosidase and glucohydrolase. They found that the enzyme activity was maximum over a pH range of 4.5 to 5.5 for both substrates. Similarly it was found earlier that maximum induction of endoglucanase was achieved at pH 5.5 (Ikram-ul-Haq et al., 2000).

Temperature and shaking: Temperature of the medium was kept at 28°C with shaking at 120 rpm, which is in accordance with the previous work conducted (Sexton et al., 2000). Esterbauer et al. (1991) found that optimum temperature for cellulase production form Trichoderma reesei was 15-28°C and optimal temperature for its growth was 30°C. Similarly Gomes et al. (1992) used Trichoderma viridie Bt 2169 for the production of cellulases and xylanases. They noted that the optimum temperature for the maximum production of these enzymes was 31.1^oC. Smith and Wood (1991) observed that the optimum temperature was 30^oC and 35^oC for the production of extracellular xylanases and β xylosidase by Aspergillus awamori.

Induction of Xylanase and Cellulase genes from different substrates: Trichoderma harzianum was grown in Vogel’s medium at 28°C for 5 days at 120 rpm for the maximal production of cellulases and xylanases with different carbon sources like CMC, xylan and glucose.

Table 1:	Xylanase and cellulase concentrations produced from T. harzianum

After 5 days the culture mixture was taken out of orbital shaker. The enzyme filtrates obtained were tested for enzyme activities. Enzyme assays were performed as described in materials and methods to check the activity of xylanases and cellulases. The results obtained are summarized in Table 1. The results are in accordance with those of earlier work conducted on Trichoderma harzianum (De Paula-Silveira et al.,1999, Emilo et al., 1996).

Present results suggest that when glucose was used as a carbon source, the production of xylanases and cellulases was inhibited and when xylan or CMC was used as a carbon source the production increased significantly. The results are in agreement with the earlier reports where it was found that XylnC production from Aspergillus nidulans was induced by xylan and repressed by glucose (MacCabe et al. (1996)). Zeilinger et al. (1996) found that xyln2 production from T. Reesei was induced by the presence of xylan and xylose.The fungus Aureobasidium pullans Y2311-1 has been reported to produce highest level of xylanase when xylan was used as a carbon source (Liang and Ljundahl, 1994). Two of endo beta –1,4 xylanases were isolated from culture filtrates of T.reesei C30 grown on xylan as a carbon source (Torronen et al., 1992).

Earlier Rajoka and Malik (1997) used cellulase, cellulosic residues, xylan, cellobiose and CMC as a carbon source for the enhanced production of cellulases by Cellulomonas strain. Ximenes et al. (1996) found that high glucose repressed the induction of cellulases whereas low glucose potentiated the enzyme production. Previously it was found by Northern analysis that genes of cellobiohydrolases were induced with 1% cellulose or CMC.

The effect of carbon sources on T.harzianum was also studied by Malik and co workers (1986). They optimized the fermentation conditions and found that maximum growth was obtained with glucose as a carbon source but negligible cellulases were produced. They suggested that substantial amounts of cellulases were produced from other carbon sources. Esterbauer et al. (1991) used CMC and xylan as a carbon source for the maximal production of glucosidase.

We, therefore, conclude that the optimum conditions for the growth of T.harzianum were at pH 5.5, temperature 28^oC and shaking at 120 rpm in Vogels medium. It was found that the xylanase and cellulase genes were induced when xylan or CMC were used as a carbon source and repressed by glucose.

ACKNOWLEDGMENTS

The authors are especially thankful to Dr. M. I. Rajoka and Dr. Farooq Latif from Natinal Institute for Biotechnology and Genetic Engineering, Faisalabad for providing Trichoderma harzianum. The work was supported by grant from Pakistan Science Foundation and Ministry of Science and Technology, Government of Pakistan.